Diabetic retinopathy is a leading cause of blindness world wide. Currently there are no therapies for the disease. Therefore, understanding mechanisms underlying the development of diabetic retinopathy will help to facilitate the development of effective therapies. Retinal cell death which occurs through an unknown high glucose-induced mechanism contributes to the disease. Nuclear accumulation of the glycolytic enzyme, glyceraldehyde-3- phosphate dehydrogenase (GAPDH), a cytosolic enzyme is an early pro-apoptotic event. Previously, our laboratory has shown that GAPDH nuclear accumulation occurs in retinal Muller (glial) cells under hyperglycemic conditions in vitro and in vivo. Since GAPDH lacks a nuclear localization signal (NLS), the exact mechanism for GAPDH nuclear accumulation is indefinite. However, recent studies have proposed a role for the NLS containing E3 ubiquitin ligase seven in absentia homolog-1 (siah-1) during lipopolysaccharide (LPS)-induced pro-apoptotic GAPDH nuclear accumulation in neurons and macrophages. These studies have suggested that IPS induced post-translational modifications of GAPDH, such as S-nitrosylation, thereby promoting GAPDH/siah-1 interaction and possible complex formation. NLS containing siah-1 then translocates in a complex with GAPDH to the nucleus. This notion is appealing since LPS is a major inducer of pro- inflammatory cytokine production, such as that of interleukin-1 p (IL-1p), and our studies have shown that IL- 1p signaling mediates high glucose-induced GAPDH nuclear accumulation in vitro. It is an intriguing idea that high glucose leads to IL-1 p mediated complex formation of GAPDH and siah-1 and translocation of this complex to the nucleus. Therefore, this project will assess (1) GAPDH/siah-1 complex formation under high glucose conditions in retinal Muller cells possibly mediated by post-translational modifications of GAPDH, and (2) whether IL-1 p mediates high glucose-induced GAPDH/siah-1 complex formation in vitro and in vivo. For theses studies cytosolic and nuclear siah-1 will be evaluated using Western blot and immunofluorescence analysis following high glucose and cytokine treatment. We will also utilize co- immunoprecipitation assays to identify GAPDH/siah-1 complex formation and proteomics analysis to test for possible post-translational modification(s) of GAPDH, which might facilitate complex formation with siah-1. We will also use diabetic caspase-1 and IL-1 receptor knock-out mice to assess the contribution of IL-1-3 to high glucose-induced GAPDH nuclear accumulation in retinal Muller cells in vivo. [unreadable] [unreadable] [unreadable]